1. Field of the Invention
This invention relates to bacterial vaccines comprising genetically-attenuated strains and to processes for their preparation.
2. Description of the Prior Art
The use of bacterial and viral vaccines has been very successful in the prevention of infectious diseases in humans and other animals. There are various methods of preparing vaccines from viruses or bacteria. The basic requirements for any vaccine and for a method for the preparation of a vaccine are that (1) the resulting vaccine contain the necessary antigenic determinants to induce formation of antibodies in the host; (2) the vaccine possess high immunogenic potential; (3) the resulting vaccine be safe enough to be administered without any danger of clinical infection, either for the recipient or any contact of the recipient, and, therefore, the risk associated with vaccination be minimized, if not totally eliminated; (4) the resulting vaccine be devoid of any toxic side-effects, for example, fever from endotoxin present in killed or extracted cells; (5) the resulting vaccine be suitable for administration by an effective route, for example, oral, intranasal, topical or parenteral; (6) the resulting vaccine and its mode of administration mimic closely the circumstances of natural infection; (7) the resulting vaccine be stable under conditions of long-term storage, and that said long-term storage be at room temperature and (8) the resulting vaccine be compatible with the usual inert vaccine carriers.
Prior art methods which have attempted to fulfill one or more of the aforementioned requirements include vaccines containing killed whole cells, purified or partially-purified selected antigenic components, and live, chemically- or genetically-attenuated microorganisms.
The use of killed whole cells as vaccines has been described by Switzer et al., in U.S. Pat. No. 4,016,253, who applied such a method to the preparation of a vaccine against Bordetella bronchiseptica infection in swine. Killed whole cells have also been used to prepare a vaccine against chronic bronchitis caused by Haemophilus influenzae (Brown and Wilson, Br. Med. J. 1:263, 1959). The use of killed cells, however, is usually accompanied by an attendant loss of immunogenic potential, since the process of killing usually destroys or alters many of the surface antigenic determinants necessary for induction of specific antibodies in the host. The antibodies produced in response to such altered antigens are not as specific for the molecular structures on the surface of the live organism, and, therefore, are not as effective against the invading pathogen.
Antigenic components, isolated and purified from microorganisms, have also been used as vaccines. This is represented, for example, by the use of purified capsular polysaccharide material of H. influenzae type b as a vaccine against the meningitis caused by this organism in humans (Parke et al., J. Inf. Dis. 136(Suppl.):S51, 1977; Anderson et al., J. Inf. Dis. 136(Suppl.):S63, 1977; Makela et al., J. Inf. Dis. 136(Suppl.):S43, 1977). This approach, however, also suffers from drawbacks, in that it requires isolation and purification techniques which may seriously compromise the detailed three dimensional spatial arrangement of the antigen upon freeing it from its position in the whole cell. The inherent immunogenicity of the antigens extracted from whole cells may also be diminished--a loss which may contribute to the relative lack of success with such vaccines in very young children (references supra; Davies, J. Immunol. 33:1, 1937 and Monto et al., J. Inf. Dis. 127:394, 1973). Thus, immunization of older children and adults with purified capsular polysaccharide from H. influenzae type b does induce humoral antibody, while the very young (less than 14-18 months) who are most susceptible to the disease fail to mount a significant, lasting response (references, supra). Similar difficulties have been encountered when purified capsular polysaccharide vaccines are prepared from Streptococcus pneumoniae and Neisseria meningitidis and used to immunize young children (Davies, J. Immunol. 33:1, 1937; Monto, et al., J. Inf. Dis. 127:394, 1973).
Chemically-attenuated, live microorganisms have also been used as vaccines in the prior art. This method of preparing vaccines is represented, for example, by the work of Wilson in U.S. Pat. Nos. 3,907,987 and 3,975,517. Wilson prepared a live, bacterial vaccine against coliform enteritis in animals from selected strains of Escherichia coli which were treated with dilute formalin in order to render them less virulent. Bauer et al., U.S. Pat. No. 4,058,599 describes the preparation of inactive but immunogenic microorganisms, which were treated with ethyleneimine and therefore rendered less infectious. The chemical treatment of whole microorganisms may severely impair their immunogenic potential, and in many cases may not decrease virulence as much as desired.
A second technique for attenuating the virulence of live microorganisms while allowing them to retain immunogenic potential, is the development of avirulent or slow-growing strains, or mutants incapable of sustained replication in the host. Such mutants, if well chosen, will maintain the full integrity of cell-surface constituents necessary for specific antibody induction, yet will be unable to cause disease, because they (1) fail to produce virulence factors, (2) grow too slowly, or (3) grow not at all in the host. A variety of such genetic variants have been used to prepare bacterial and viral vaccines.
Smith (U.S. Pat. No. 3,364,117), Hillman (U.S. Pat. No. 4,133,875) and Germanier (U.S. Pat. No. 3,856,935) have all described vaccine strains which have lost the ability to cause disease presumably because of mutations in genes responsible for the production of virulence factors. The vaccine described by Smith (supra) comprised a "rough" variant of Salmonella cholerasuis which was poorly characterized, except for its loss of virulence in pigs. Since the mutant is rough, the surface properties are altered, and, therefore, not truly representative of the antigens of the pathogenic form. Hillman (supra) described a well-characterized mutant strain of Streptococcus mutans which has a single point mutation in the structural gene that codes for the enzyme L(+)-lactate dehydrogenase. This mutant strain is a low-acid producer and potentially can replace the normal high-acid producing Streptococcus mutans in oral flora in order to reduce the incidence and severity of dental caries in humans. Germanier (supra) described a mutant strain of Salmonella typhi carrying a single mutation (characterized as a deletion) which profoundly affects the expression of the genes encoding the galactose-metabolising enzymes. These genes are clustered in a unit, the gal operon, which encodes the information for the synthesis of three enzymes--uridine diphosphogalactose-4-epimerase, galactose-1-phosphate uridylyltransferase and galactokinase. The deletion in this strain has occurred in the epimerase gene which prevents the production of any functional epimerase enzyme. Characteristically such a deletion also exerts strong polar effects on the distal genes of the operon (Martin et al., Cold Spring Harbor Symp. Quant. Biol. 31:215, 1966) hence the levels of galactokinase and galactose-1-phosphate uridylyl-transferase are markedly reduced in this strain. Deletions are usually stable and no revertants of this strain have been detected. However, such galE mutations cause alterations in surface properties by preventing the complete formation of the lipopolysaccharide side chains. Hence, the very structures responsible for inducing type-specific antibodies are compromised. In addition, galE mutations can result in autolysis, thereby compromising the ability of the vaccine strain to produce prolonged immunological stimulation.
Influenza and respiratory syncytial virus mutant strains which replicate slowly at human body temperature (37.degree. C.) and thus are unable to cause disease, have been prepared (Murphy et al., J. Inf. Dis. 128:479, 1973 and Chanock et al., Pediatr. Res. 11:264, 1977). These single mutation strains have proven to produce so many virulent revertants in the vaccinee that they are not practical. There are no reports of slow-growing bacterial mutant strains appropriate for use as vaccines, but the same limitations which apply to the slow-growing viral strains would also apply to them.
Two classes of mutants have been used to prepare vaccine strains which do not replicate at all in the host. The first class includes strains which are dependent upon unusual nutrients or growth factors (compounds outside the biochemistry of the vaccinee) for replication as described by Levine (J. Inf. Dis. 133:424, 1976) and Reitman (J. Inf. Dis. 117:101, 1967). These workers described strains of Salmonella typhi and Salmonella typhosa which require streptomycin for growth. Presumably the structure of ribosomal proteins in these strains is modified so that streptomycin is required for proper ribosome function. These strains, however, do not have sufficient genetic stability to make them truly safe vaccines.
The second class includes strains which are incapable of replication above certain temperatures as described by Fahey and Cooper (Infect. Immun. 1:263, 1970), Maheswaran (U.S. Pat. No. 3,855,408) and Helms et al., (J. Inf. Dis. 135:582, 1977). Temperature-sensitive strains of Salmonella enteritidis, Pasteurella multocida and Streptococcus pneumoniae were isolated and partially-characterized by these workers. The strains were unable to grow at the body-temperature of the animals they were tested in, but were excellent immunogens, since the surface properties of the cells were unaltered. Significant reversion to virulence, however, has precluded their use in humans and other animals.
The reversion problem associated with attempts to use temperature-sensitive strains as vaccines appears to be an inherent limitation of the approach. This is because ts mutations result from single base changes and therefore exhibit significant spontaneous reversion. Similarly, reversion (to virulence) has also posed a problem in the safety of employing genetically-attenuated viral strains as vaccines (Henderson et al., J.A.M.A. 190:41, 1964; Chanock et al., Pediatr. Res. 11:264, 1977 and Murphy et al., J. Inf. Dis. 128:479, 1973)
In order to overcome this problem, the prior art has investigated the vaccine potential of virus strains which contain two growth-attenuating mutations (Murphy et al., Virology 88:231, 1978 and Murphy et al., Infect. Immun. 23:249, 1979). In such strains, two mutations of different phenotype were combined with the expectation that the reversion frequencies would decrease.
Two methods have been used to combine multiple ts mutations in one strain. First, Chanock et al., (Pediatr. Res. 11:264, 1977) mutagenized a strain of respiratory syncytial virus which already contained a ts mutation, and isolated a strain which contained a second ts lesion. The first mutation almost completely blocked virus replication at 39.degree. C.; the second mutation severely limited replication at 37.degree. C., and so the double mutant strain could be recognized.
A second method using natural in vivo recombination was reported by Murphy et al., (Virology 88:231, 1978). They constructed a doubly ts strain of influenza virus by co-infecting cells with two virus strains, one containing a mutation with a cut-off temperature of 37.degree. C. and the second containing a mutation in another gene with a cut-off temperature of 38.degree. C., and isolating recombinant viruses among the progeny. The rationale for preparation of these strains was that the resulting isolates would contain two growth restricting mutations, the original mutation which severely restricts replication at 38.degree. or 39.degree. C. and a second mutation which severely restricts replication at or above 37.degree. C. At temperatures of 39.degree. C. or above both mutations severely limit virus replication; and revertants are extremely rare (as outlined above). However, at 37.degree. C., normal human body temperature, only one of the two mutations will limit growth and the virus will phenotypically express only one mutation; resulting in a reversion frequency equal to that of the 37.degree. C. mutation alone. Double-ts mutant strains, where the temperature cut-off points are not the same, can also produce significant numbers of revertants of one of the ts mutations if they are propagated at temperatures near the lower cut-off temperature. Such temperatures are semi-permissive and provide strong selective pressure for overgrowth of the original strain by revertants.
While the use of multiple mutations has not been applied to bacterial vaccine strain preparation, Curtiss has constructed a strain of Escherichia coli which is attenuated by the incorporation of multiple mutations of different phenotype, for gene cloning (Ann. Rev. Microbiol. 30:507, 1976). The important consideration in this work was to produce a strain that could not survive outside a specialized environment provided in the laboratory. In fact, at least one of the mutations introduced into the strain causes the self-destruction of the cell when it attempts to grow outside the defined specialized environment. Curtiss has suggested that this strain could be used to clone the gene(s) for a bacterial toxin which could then be produced in bulk for vaccine use (ibid, at 510, lines 26-28). Furthermore, British patent specification No. 1-516-458, published by July 5, 1978 in the United Kingdom, describes this microorganism, Escherichia coli K-12.sub.x 1776 and the many mutations which have been introduced into it in order to make it safe for the large-scale production of prokaryotic or eukaryotic proteins (for example, cholera toxin or insulin), or organic acids (for example, succinic or lactic acid produced by fermentation of glucose). The specification describes on p. 4 the use of microorganisms which have been precluded from growth or colonization in natural ecological niches. However, the main thrust of the specification is directed towards the construction of bacterial strains containing various mutations conveying many different phenotypes in one strain. The disadvantage of using such an approach for construction of vaccine strains are discussed below.
The combination of two mutations of different phenotype in a single strain will only be effective in reducing the revertant frequency when both mutations are simultaneously restrictive. This argument is equally applicable to temperature-sensitive, growth factor or virulence factor mutations, alone or in combination. Ideally, to achieve reduced reversion frequencies by combining two or more mutations in a single strain, the mutations must convey identical phenotypes so as to ensure that they always work in concert.